International Immunology, Vol 2, No. 7
© 1990 Oxford University Press 0953-8178/90 $3 00
Molecular cloning of a cDNA encoding the human interleukin 4 receptor Jean-Pierre Galizzi, Caroline E. Zuber, Nobuyuki Harada1, Daniel M. Gorman2, Odile Djossou, Robert Kastelein2, Jacques Banchereau, Maureen Howard1, and Atsushl Miyajima2 Schering-Plough, 27 chemin des Peuphers, BP11, 69572 Dardilly, France Departments of immunology and 2Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304, USA Key words cytokine, cytokine receptor, gene family, B cell stimulating factor
Using the mouse interleukin 4 (IL-4) receptor cDNA as a probe, we Isolated a cDNA encoding the human IL-4 receptor (hlL-4 receptor) from a multlfactor-responsive human myeloid cell line, TF1. The cDNA encodes for an open reading frame of 825 amino acids Including a signal sequence (25 amlno acids), the external domain (207 amlno acids), a transmembrane domain (24 amlno acids), and a large cytoplasmlc domain (569 amino acids). The human IL-4 receptor has a 65% identity with the mouse IL-4 receptor at the nucleic acid level and retains the typical structural motif of the previously described cytokine receptor family. COS7 cells transfected with the fulllength cDNA expressed high levels (140,000 sites/cell) of IL-4 binding sites, with a Kd = 80 pM, an affinity Identical to that of the original TF1 cells. Similar to IL-4 responsive cells, cross-linking of [i23|]IL-4 to COS7 cells transfected with the cDNA showed a major protein of 130-150 kd and minor species of 5 5 - 8 5 kd.
Introduction Human interleukin 4 (hlL-4) was initially identified as a recombinant protein derived from a cDNA homologous to mouse IL-4 (1); it exhibits the same biological properties as mouse IL-4 (2,3). Briefly, hlL-4 is produced by T cells and acts as a growth factor for pre-activated B cells (4) and T cells (5). It acts on enriched B cell populations to produce IgE (6) and on purified B cells to secrete IgG and IgM (7). It enhances the generation of cytotoxic T cells but inhibits the IL-2-dependent generation of lymphocyteactivated killer cells (8,9). hlL-4 shows numerous growth and differentiation promoting effects on other hemopoietic lineages (for a review see 10). IL-4 elicits its biological activities by binding to specific receptors on the cell surface of IL-4-responsive cells (11,12). Binding of IL-4 to its receptor causes rapid receptor internalization followed by up-regulation of IL-4 receptor expression in the case of a human B lymphoma (13), human tonsillar B cells (14), and mouse T and B cells (15). Thus, up-regulation of the IL-4 receptor by its ligand may be important for its biological activity. Recently a cDNA encoding the mouse IL-4 receptor was isolated from mast cells by direct expression in COS7 cells (16) and from T cells using a subtracted cDNA probe derived from
a CTLL cell variant expressing a large number of IL-4 receptors (17). The mouse IL-4 receptor cDNA encoded a protein of 810 amino acid residues and expressed a 140 kd protein in COS7 cells which bound IL-4 with high affinity The external domain of the IL-4 receptor had a structure typical of members of the recently described family of cytokine receptors, which includes the IL-2 receptor /3 chain (18) and the IL-3 (19), IL-6 (20), GM-CSF (21), and erythropoietin (22) receptors. Although mouse IL-4 induced protein tyrosine phosphorylation in hemopoietic cells (23), the cytoplasmic domain of the cloned IL-4 receptor did not have any consensus sequence of kinases (16,17). The cytoplasmic domain lacked significant sequence homology with other receptors but contained many prolines and serines, which are also abundant in the cytoplasmic domains of the IL-3 receptor (19) and the IL-2 receptor /S chain (18). We previously purified a human IL-4 binding protein from the gibbon T cell line, MLA 144 (24). Consistent with the cloned mouse IL-4 receptor, the IL-4 receptor purified from detergentsolubilized extracts had a mol. wt of 130-140 kd and bound IL-4 with high affinity. Here we describe the cloning and expression of the human IL-4 receptor cDNA.
Correspondence to: Atsushi Miyajima, as above Transmitting editor. K Ishizaka
Received 20 March 1990, accepted 23 April 1990
Downloaded from http://intimm.oxfordjournals.org/ at UQ Library on November 23, 2014
Abstract
670
Human IL-4 receptor
Methods Cytokines Purified hlL-4 and human granulocyte-macrophage colony stimulating factor (GM-CSF) derived from Eschenchia coli were provided by Schering-Plough Research (Bloomfield, NJ) For binding assays hlL-4 was lodinated to a specific radioactivity of 2500 Ci/mol with iodogen (Pierce), as described previously (16). Cell lines and culture
Construction of cDNA libraries Poly(A)+ RNA was prepared by the guanidine thiocyanate method (26) or using the Fast Track mRNA isolation kit (Invitrogen, CA) RNA was converted to double-stranded cDNA and SsfXI adaptors (Invitrogen) were attached to the cDNA. cDNA larger than 2 0 kb were isolated by electrophoresis on a 0.8% agarose gel. The cDNA library of the Burkitt lymphoma, BL41, was constructed by inserting the size-selected cDNA into the SsfXI sites of the pCEV4 vector (19), a derivative of the pcDSRa cDNA expression vector (27), and 1.2x10 6 independent clones were obtained A phage cDNA library of TF1 was constructed by using the oligo(dT) primer containing the Not\ restriction site and the EcoRI adaptor according to the protocol provided by Promega (Madison, Wl)
Transfection of COS7 cells COS7 cells were transfected with cDNA by electroporation Exponentially growing COS7 cells were detached by trypsin and washed with RPMI 1640. Cells were suspended in RPMI 1640 (8X10 6 cells/0.5 ml) and incubated with 18^g of DNA for 10 mm at room temperature. Electroporation was carried out in a 0.4 cm cuvett at 960 /iF and 220 V. Cells were grown in RPMI 1640 medium containing 10% FCS for 3 days before binding assays. Binding assays COS7 cells were detached from the plates by a short incubation in PBS containing 0.5 mM EDTA. Exponentially growing TF1 cells were harvested by centrifugation Cells were washed and resuspended in RPMI 1640 containing 20 mM HEPES, 0.5% BSA. Cells (7x1CH COS7 cells or 1 2x106 TF1 cells) were incubated with 5 - 2 5 0 p M of [125l]IL-4 in the presence or absence of 10 nM nonradioactive IL-4 at 4°C for 3 h. To prevent cell loss during washing, 106 mouse MC/9 cells, which do not bind human IL-4, were added to each sample at the end of the incubation. Cell-bound radioactivity was separated by centrifugation as described previously (13) The affinity of IL-4 binding to its receptor and the number of IL-4 receptors per cell were estimated by Scatchard plot analysis of equilibrium binding data using the LIGAND program Cross-linking of Cells (2x106 COS7, 10x10 6 Raji, or 5x10« TF1 cells) were incubated with 150 pM [125l]IL-4 in RPMI 1640 containing 20 mM HEPES (pH 7 3) at 4°C After a 2 h incubation cells were
Screening of cDNA libraries The BL41 cDNA library was screened by colony hybridization using the mouse IL-4 receptor cDNA as a probe (2.5 kb fragment encoding the entire mouse IL-4 receptor). Colonies were transferred to nylon membrane filters (Schleicher & Schuell), and hybridization was performed at 42°C in 6 x SSPE (1 x SSPE contains: 17.4 g/l NaCI, 2.76 g/1 NaH2PO4.H2O, 7.4 g/l EDTA, pH 7.4) containing 20% formamide, 5xDenhardt's solution [1 xDenhardt's solution: 100mg/l Ficoll, 100mg/l polyvinylpyrrolidone (mean mol. wt = 360,000), 100 mg/l BSA (pentax fraction V)], 0 . 1 % sodium dodecylsulfate, and 100 /ig/ml E coli tRNA. Filters were washed with 4 x SSPE solution at 50°C and exposed to X-ray films. To isolate a full-length cDNA we used the DNA fragment (the 5' end to the Nco\ site) from the 4A2 cDNA clone isolated from the BL41 cDNA library as a probe. Hybridization was performed in solutions containing 50% formamide at 42 °C and filters were washed at 60°C with 0 2 x SSPE RNA blot analysis Two micrograms of poly(A)+ RNA were denatured and fractionated by electrophoresis on a 2% agarose gel containing formaldehyde. RNA was transferred to a nylon membrane filter (Schleicher & Schuell) and cross-linked by UV irradiation in a Stratalinker (Stratagen). The filter was hybridized for 24 h with
1 2 3 28 S-
18S-
Fig. 1. RNA Wot analysis of the hlL-4 receptor mRNA Two micrograms of po)y(A)+ RNA prepared from BL41 (lane 1), Daudi (lane 2), and PHA blasts cultured for 24 h in the presence of IL-4 (lane 3) was denatured by formaldehyde and formamide and fractionated by electropnoresis on an agarose gel RNA was transferred to nyton membranes and hybridized wrth the 1.9 kb fragment of 4A2 cDNA (5' end to Nco\) The position of the 28S and 18S nbosomal RNA are indicated.
Downloaded from http://intimm.oxfordjournals.org/ at UQ Library on November 23, 2014
The human Burkitt lymphoma cell line, BL41, was provided by Dr G. Lenoir (International Agency for Cancer, Lyon, France) Daudi and Raji cells were obtained from ATCC Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, streptomycin (50 /xg/ml), and penicillin (100 Ill/ml). The human multifactor-dependent myeloid cell line, TF1 (25), was provided by Dr T. Kitamura (DNAX). TF1 cells were cultured in RPM11640 supplemented with 10% FCS and 1 ng/ml human GM-CSF. COS7 cells were cultured in RPM11640 medium containing 10% FCS.
.m./ml of the 1.9 kb human IL-4 receptor probe (the 5' end to the Nco\ fragment) after a 3 h prehybndization Finally, the filter was washed in 0 1 xSSPE at 65°C for 90 mm and exposed to an X-ray film for 3 days.
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© 1990 Oxford University Press 0953-8178/90 $3 00
Molecular cloning of a cDNA encoding the human interleukin 4 receptor Jean-Pierre Galizzi, Caroline E. Zuber, Nobuyuki Harada1, Daniel M. Gorman2, Odile Djossou, Robert Kastelein2, Jacques Banchereau, Maureen Howard1, and Atsushl Miyajima2 Schering-Plough, 27 chemin des Peuphers, BP11, 69572 Dardilly, France Departments of immunology and 2Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304, USA Key words cytokine, cytokine receptor, gene family, B cell stimulating factor
Using the mouse interleukin 4 (IL-4) receptor cDNA as a probe, we Isolated a cDNA encoding the human IL-4 receptor (hlL-4 receptor) from a multlfactor-responsive human myeloid cell line, TF1. The cDNA encodes for an open reading frame of 825 amino acids Including a signal sequence (25 amlno acids), the external domain (207 amlno acids), a transmembrane domain (24 amlno acids), and a large cytoplasmlc domain (569 amino acids). The human IL-4 receptor has a 65% identity with the mouse IL-4 receptor at the nucleic acid level and retains the typical structural motif of the previously described cytokine receptor family. COS7 cells transfected with the fulllength cDNA expressed high levels (140,000 sites/cell) of IL-4 binding sites, with a Kd = 80 pM, an affinity Identical to that of the original TF1 cells. Similar to IL-4 responsive cells, cross-linking of [i23|]IL-4 to COS7 cells transfected with the cDNA showed a major protein of 130-150 kd and minor species of 5 5 - 8 5 kd.
Introduction Human interleukin 4 (hlL-4) was initially identified as a recombinant protein derived from a cDNA homologous to mouse IL-4 (1); it exhibits the same biological properties as mouse IL-4 (2,3). Briefly, hlL-4 is produced by T cells and acts as a growth factor for pre-activated B cells (4) and T cells (5). It acts on enriched B cell populations to produce IgE (6) and on purified B cells to secrete IgG and IgM (7). It enhances the generation of cytotoxic T cells but inhibits the IL-2-dependent generation of lymphocyteactivated killer cells (8,9). hlL-4 shows numerous growth and differentiation promoting effects on other hemopoietic lineages (for a review see 10). IL-4 elicits its biological activities by binding to specific receptors on the cell surface of IL-4-responsive cells (11,12). Binding of IL-4 to its receptor causes rapid receptor internalization followed by up-regulation of IL-4 receptor expression in the case of a human B lymphoma (13), human tonsillar B cells (14), and mouse T and B cells (15). Thus, up-regulation of the IL-4 receptor by its ligand may be important for its biological activity. Recently a cDNA encoding the mouse IL-4 receptor was isolated from mast cells by direct expression in COS7 cells (16) and from T cells using a subtracted cDNA probe derived from
a CTLL cell variant expressing a large number of IL-4 receptors (17). The mouse IL-4 receptor cDNA encoded a protein of 810 amino acid residues and expressed a 140 kd protein in COS7 cells which bound IL-4 with high affinity The external domain of the IL-4 receptor had a structure typical of members of the recently described family of cytokine receptors, which includes the IL-2 receptor /3 chain (18) and the IL-3 (19), IL-6 (20), GM-CSF (21), and erythropoietin (22) receptors. Although mouse IL-4 induced protein tyrosine phosphorylation in hemopoietic cells (23), the cytoplasmic domain of the cloned IL-4 receptor did not have any consensus sequence of kinases (16,17). The cytoplasmic domain lacked significant sequence homology with other receptors but contained many prolines and serines, which are also abundant in the cytoplasmic domains of the IL-3 receptor (19) and the IL-2 receptor /S chain (18). We previously purified a human IL-4 binding protein from the gibbon T cell line, MLA 144 (24). Consistent with the cloned mouse IL-4 receptor, the IL-4 receptor purified from detergentsolubilized extracts had a mol. wt of 130-140 kd and bound IL-4 with high affinity. Here we describe the cloning and expression of the human IL-4 receptor cDNA.
Correspondence to: Atsushi Miyajima, as above Transmitting editor. K Ishizaka
Received 20 March 1990, accepted 23 April 1990
Downloaded from http://intimm.oxfordjournals.org/ at UQ Library on November 23, 2014
Abstract
670
Human IL-4 receptor
Methods Cytokines Purified hlL-4 and human granulocyte-macrophage colony stimulating factor (GM-CSF) derived from Eschenchia coli were provided by Schering-Plough Research (Bloomfield, NJ) For binding assays hlL-4 was lodinated to a specific radioactivity of 2500 Ci/mol with iodogen (Pierce), as described previously (16). Cell lines and culture
Construction of cDNA libraries Poly(A)+ RNA was prepared by the guanidine thiocyanate method (26) or using the Fast Track mRNA isolation kit (Invitrogen, CA) RNA was converted to double-stranded cDNA and SsfXI adaptors (Invitrogen) were attached to the cDNA. cDNA larger than 2 0 kb were isolated by electrophoresis on a 0.8% agarose gel. The cDNA library of the Burkitt lymphoma, BL41, was constructed by inserting the size-selected cDNA into the SsfXI sites of the pCEV4 vector (19), a derivative of the pcDSRa cDNA expression vector (27), and 1.2x10 6 independent clones were obtained A phage cDNA library of TF1 was constructed by using the oligo(dT) primer containing the Not\ restriction site and the EcoRI adaptor according to the protocol provided by Promega (Madison, Wl)
Transfection of COS7 cells COS7 cells were transfected with cDNA by electroporation Exponentially growing COS7 cells were detached by trypsin and washed with RPMI 1640. Cells were suspended in RPMI 1640 (8X10 6 cells/0.5 ml) and incubated with 18^g of DNA for 10 mm at room temperature. Electroporation was carried out in a 0.4 cm cuvett at 960 /iF and 220 V. Cells were grown in RPMI 1640 medium containing 10% FCS for 3 days before binding assays. Binding assays COS7 cells were detached from the plates by a short incubation in PBS containing 0.5 mM EDTA. Exponentially growing TF1 cells were harvested by centrifugation Cells were washed and resuspended in RPMI 1640 containing 20 mM HEPES, 0.5% BSA. Cells (7x1CH COS7 cells or 1 2x106 TF1 cells) were incubated with 5 - 2 5 0 p M of [125l]IL-4 in the presence or absence of 10 nM nonradioactive IL-4 at 4°C for 3 h. To prevent cell loss during washing, 106 mouse MC/9 cells, which do not bind human IL-4, were added to each sample at the end of the incubation. Cell-bound radioactivity was separated by centrifugation as described previously (13) The affinity of IL-4 binding to its receptor and the number of IL-4 receptors per cell were estimated by Scatchard plot analysis of equilibrium binding data using the LIGAND program Cross-linking of Cells (2x106 COS7, 10x10 6 Raji, or 5x10« TF1 cells) were incubated with 150 pM [125l]IL-4 in RPMI 1640 containing 20 mM HEPES (pH 7 3) at 4°C After a 2 h incubation cells were
Screening of cDNA libraries The BL41 cDNA library was screened by colony hybridization using the mouse IL-4 receptor cDNA as a probe (2.5 kb fragment encoding the entire mouse IL-4 receptor). Colonies were transferred to nylon membrane filters (Schleicher & Schuell), and hybridization was performed at 42°C in 6 x SSPE (1 x SSPE contains: 17.4 g/l NaCI, 2.76 g/1 NaH2PO4.H2O, 7.4 g/l EDTA, pH 7.4) containing 20% formamide, 5xDenhardt's solution [1 xDenhardt's solution: 100mg/l Ficoll, 100mg/l polyvinylpyrrolidone (mean mol. wt = 360,000), 100 mg/l BSA (pentax fraction V)], 0 . 1 % sodium dodecylsulfate, and 100 /ig/ml E coli tRNA. Filters were washed with 4 x SSPE solution at 50°C and exposed to X-ray films. To isolate a full-length cDNA we used the DNA fragment (the 5' end to the Nco\ site) from the 4A2 cDNA clone isolated from the BL41 cDNA library as a probe. Hybridization was performed in solutions containing 50% formamide at 42 °C and filters were washed at 60°C with 0 2 x SSPE RNA blot analysis Two micrograms of poly(A)+ RNA were denatured and fractionated by electrophoresis on a 2% agarose gel containing formaldehyde. RNA was transferred to a nylon membrane filter (Schleicher & Schuell) and cross-linked by UV irradiation in a Stratalinker (Stratagen). The filter was hybridized for 24 h with
1 2 3 28 S-
18S-
Fig. 1. RNA Wot analysis of the hlL-4 receptor mRNA Two micrograms of po)y(A)+ RNA prepared from BL41 (lane 1), Daudi (lane 2), and PHA blasts cultured for 24 h in the presence of IL-4 (lane 3) was denatured by formaldehyde and formamide and fractionated by electropnoresis on an agarose gel RNA was transferred to nyton membranes and hybridized wrth the 1.9 kb fragment of 4A2 cDNA (5' end to Nco\) The position of the 28S and 18S nbosomal RNA are indicated.
Downloaded from http://intimm.oxfordjournals.org/ at UQ Library on November 23, 2014
The human Burkitt lymphoma cell line, BL41, was provided by Dr G. Lenoir (International Agency for Cancer, Lyon, France) Daudi and Raji cells were obtained from ATCC Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, streptomycin (50 /xg/ml), and penicillin (100 Ill/ml). The human multifactor-dependent myeloid cell line, TF1 (25), was provided by Dr T. Kitamura (DNAX). TF1 cells were cultured in RPM11640 supplemented with 10% FCS and 1 ng/ml human GM-CSF. COS7 cells were cultured in RPM11640 medium containing 10% FCS.
.m./ml of the 1.9 kb human IL-4 receptor probe (the 5' end to the Nco\ fragment) after a 3 h prehybndization Finally, the filter was washed in 0 1 xSSPE at 65°C for 90 mm and exposed to an X-ray film for 3 days.
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